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Objectives: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK)., Procedure: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined., Results: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples., Conclusion: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas., (© 2023 American College of Veterinary Ophthalmologists.)

Objectives: To identify the minimum inhibitory concentration (MIC) distribution for commonly used topical antibiotics from isolates of dogs and horses with ulcerative bacterial keratitis, and to investigate changes in MIC values over time and following treatment with topical fluoroquinolones., Animals Studied: One hundred thirty-four client-owned dogs and 20 client-owned horses with bacterial ulcerative keratitis., Procedure: Minimum inhibitory concentration values for 14 topical antibiotics were reported for canine and equine cases of bacterial ulcerative keratitis between 2013 and 2018. Changes in MIC values over time and after treatment with topical fluoroquinolones were reported., Results: The three most common bacterial genera isolated were Staphylococcus, Streptococcus, and Pseudomonas. Together, these represented 79.4% of canine cases and 77.4% of equine cases. Overall, isolates from horses tended to have lower MIC values, as did Pseudomonas isolates from both dogs and horses, compared to other bacterial genera, especially Staphylococcus spp. The MIC values of erythromycin and trimethoprim sulfa for Staphylococcus spp., and the MIC value of moxifloxacin for Pseudomonas significantly increased over time. Previous topical fluoroquinolone use was associated with a significant increase in the MIC value of ofloxacin in canine and equine Staphylococcus isolates and current topical fluoroquinolone use was associated with significant increases in the MIC values of ciprofloxacin, moxifloxacin, and ofloxacin in canine Staphylococcus isolates., Conclusion: Patients previously or currently treated with topical fluoroquinolones, particularly in Staphylococcus infections, may require alternative antibiotics or additional antibiotic classes other than fluoroquinolones. Bacterial culture with MIC susceptibility testing should be highly recommended when a Staphylococcal infection is suspected., (© 2020 American College of Veterinary Ophthalmologists.)

Objective: Heat-shock proteins, particularly the 70-kDa member (Hsp70), have been implicated in facilitating wound healing in multiple tissues. Expression and localization of three HSPs were assessed in normal and wounded canine corneas to elucidate a role in epithelial healing., Methods: Paraffin-embedded normal corneas, acute and repeatedly abraded corneas, and keratectomies of spontaneous chronic corneal epithelial defects (SCCEDs) were subjected to routine immunohistochemistry for Hsp27, 47, and 70 expression. Ex vivo corneal defects were created and treated with anti-HSPs or IgG controls, and wound healing was monitored. Primary cultures of canine corneal stromal fibroblasts and corneal epithelial cells were treated with exogenous Hsp70, and an artificial wound was created in vitro to monitor restoration of the monolayer., Results: Normal canine corneas exhibited constitutive expression of all HSPs evaluated. Inducible expression was demonstrated in acutely wounded tissues, and expression in the chronically abraded corneas was relocalized. All HSP expression was below the limits of detection in the epithelium of SCCED samples. Inhibition of HSPs in culture resulted in delayed wound healing when compared to controls. Hsp70-treated fibroblasts demonstrated significantly (P < 0.001) increased migration and proliferation compared to the vehicle control; however, there was no significant effect of exogenous Hsp70 on corneal epithelial cells., Conclusions: These findings suggest that HSPs are induced in the normal canine cornea during re-epithelialization. Hsp70 expression is likely important for inducing the cytoarchitectural remodeling, migration, and proliferation necessary early in the canine corneal healing response, and suppressed expression may contribute to the pathophysiology of nonhealing defects., (© 2015 American College of Veterinary Ophthalmologists.)

Objective: To evaluate effect of adjunctive treatment with tetracycline analogues on time to complete corneal reepithelialization in dogs with nonhealing (ie, refractory) corneal ulcers., Design: Randomized controlled clinical trial., Animals: 89 dogs with refractory corneal ulcers., Procedures: Corneal ulcers were treated via debridement and grid keratotomy. Dogs were assigned to receive 1 of 3 treatment regimens for up to 6 weeks: doxycycline (5 mg/kg [2.27 mg/lb], PO, q 12 h) with topically applied ophthalmic ointment containing neomycin, polymyxin B, and bacitracin (ie, triple antibiotic ointment; q 8 h); cephalexin (22 mg/kg [10 mg/lb], PO, q 12 h) with topically applied oxytetracycline ophthalmic ointment (q 8 h); or a control treatment of cephalexin (22 mg/kg, PO, q 12 h) with topically applied triple antibiotic ointment (q 8 h). Healing was monitored via measurements of the wound with calipers and evaluation of photographs obtained every 2 weeks. Treatment effectiveness was evaluated by wound healing and decreased signs of pain., Results: The Boxer breed was overrepresented in all groups. At the 2-week time point, wound healing was significantly more common in small-breed dogs, compared with large-breed dogs. Dogs treated with oxytetracycline ophthalmic ointment had a significantly shorter healing time than did dogs receiving the control treatment. Corneal ulcers in dogs that received doxycycline PO healed more rapidly than did ulcers in dogs in the control treatment group; however, this difference was not significant., Conclusions and Clinical Relevance: Topical tetracycline ophthalmic ointment was a safe, inexpensive, and effective adjunctive treatment for refractory corneal ulcers in dogs.

Purpose: To determine if molecular markers typically associated with ultraviolet exposure could be detected in canine ocular hemangiomas (HA) and hemangiosarcomas (HSA)., Methods: Paraffin-embedded samples of canine ocular HA (n = 6) and HSA (n = 6) were examined for the presence of p53, p21, p16, cyclin D, PCNA, pAkt, telomerase, and estrogen receptor (ER)-alpha using immunohistochemistry., Results: p53 and cyclin D protein were not detected in any of the canine HA or HSA samples. The majority of the HA and HSA were negative for both p21 and telomerase. pAkt immunoreactivity was absent in one HA, one HSA, but was present in five HA and five HSA. All of the HA or HSA samples were strongly positive for p16 and PCNA. ERalpha was expressed in all of the samples examined; there was more intense staining in the HSA samples compared to the HA samples., Conclusions: Results from this study describe the protein expression, via immunohistochemistry, that might be altered in UV exposure in HA and HAS formation. p53 may not play an important role in tumor development; rather, in the tumors examined, expression of cell cycle regulators independent of the p53 pathway appear central in HA and HSA formation and progression. In addition, this study finds that ERalpha may be involved in promoting the invasive behavior associated with HSA.

Purpose: To determine if cyclooxygenase-2 (COX-2) is upregulated when lens epithelial cells (LEC) in clinical samples of cataracts and posterior capsule opacification (PCO) undergo epithelial-mesenchymal transition (EMT)-like changes. We also wanted to learn if inhibition of the enzymatic activity of COX-2 could prevent PCO formation., Methods: To ensure that EMT-like changes were occurring in LEC, real-time RT-PCR was used to examine expression of EMT markers. Clinical samples of canine cataracts and PCO were examined for COX-2 expression using immunohistochemistry, western blot analysis, and real-time RT-PCR. The COX-2 inhibitors, rofecoxib and celecoxib, were used in an ex vivo model of PCO formation, and the effects on cellular migration, proliferation, and apoptosis were analyzed using immunohistochemistry and western blots. Prostaglandin E2 (PGE2) expression was examined with ELISA., Results: Markers of EMT, such as lumican, Snail, Slug, and COX-2 were expressed in LEC. In clinical samples of cataracts and PCO, there was overexpression of COX-2 protein and mRNA. Both rofecoxib and celecoxib were effective at inhibiting PCO formation in our ex vivo model. Prevention of PCO with the COX-2 inhibitors appeared to work through decreased migration and proliferation, and increased apoptosis. Neither of the drugs had a toxic effect on confluent LEC and appeared to inhibit PCO through their pharmacologic action. Synthesis of PGE2 was inhibiting in the capsules treated with the COX-2 inhibiting drugs., Conclusions: Extracapsular phacoemulsification cataract surgery is the most common surgical procedure performed in human and veterinary ophthalmology. The most frequent postoperative complication is PCO. The LEC that remain adhered to the lens capsule undergo EMT-like changes, proliferate, and migrate across the posterior lens capsule causing opacities. We have shown that COX-2, a protein associated with EMT, is upregulated in canine cataracts and PCO. Inhibiting the enzymatic activity effectively prevented EMT of LEC in our ex vivo model of PCO through pharmacologic action, and not acute toxicity. These findings indicate that using COX-2 inhibitors in vivo may be an effective technique in preventing PCO.

Objective Compare the amount and distribution of pigment in normal canine eyes with blue and brown irides. Animal studied Eighteen mixed-breed dogs euthanized for population control. Procedures Intact globes were removed immediately following euthanasia. Iris color was noted, and globes were evaluated histologically to determine the distribution of pigment. High magnification (×400) digital images were taken of the dorsal and ventral ciliary processes (CP) of 13 eyes with blue irides and 13 eyes with brown irides. Low magnification (×20) images were taken of the dorsal and ventral portions of the ciliary body (CB) of 14 eyes with blue irides and 14 eyes with brown irides. The images were digitally analyzed to determine the percent of the CP and CB that were pigmented in the high and low magnification images, respectively. Results Eyes with brown irides contained abundant melanin pigment around the CB musculature. This pigment was absent in eyes with blue irides, and the difference was statistically significant ( P = 0.001) when digitally analyzed using the low magnification images. Iris color did not have a significant ( P = 0.34) impact on the amount of melanin within the CP, as determined using the high magnification images. Conclusions Compared to eyes with brown irides, eyes with blue irides lack pigment around the CB musculature, but contain comparable amounts of pigment in the CP. [ABSTRACT FROM AUTHOR]

Objective--To evaluate whether the effects of oxidative stress could be attenuated in cultures of canine lens epithelial cells (LECs) by incubation with grape seed proanthocyanidin extract (GSE), resveratrol (RES), or a combination of both (GSE+RES). Sample Population--Primary cultures of canine LECs. Procedures--LECs were exposed to 100µM tertiary butyl-hydroperoxide (TBHP) with or without GSE, RES, or GSE+RES. The dichlorofluorescein assay was used to detect production of reactive oxygen species (ROS), and immunoblot analysis was used to evaluate the expression of stress-induced cell-signaling markers (ie, the mitogen-activated protein kinase [MAPK] and phosphoinositide-3 kinase [PI3K] pathways). Results--GSE and GSE+RES significantly reduced ROS production after a 30-minute exposure to TBHP. Only GSE significantly reduced ROS production after a 120-minute exposure to TBHR Incubation with GSE reduced TBHP-induced activity of the MAPK and PI3K pathways. Conclusions and Clinical Relevance--GSE inhibited key components associated with cataractogenesis, ROS production, and stress-induced cell signaling. On the basis of the data reported here, there is strong evidence that GSE could potentially protect LECs from the damaging effects of oxidative stress. [ABSTRACT FROM AUTHOR]